The non-catalytic function of PARP2 in DNA repair and cancer therapy

PARP2在DNA修复和癌症治疗中的非催化功能

• 批准号: 10540084
• 负责人: Shan Zha
• 金额: $ 59.71万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10540084
• 关键词: ADP Ribose Transferases; ADP ribosylation; Acute Myelocytic Leukemia; Address; Adult; Affect; Affinity; Anemia; Animals; BRCA deficient; BRCA1 gene; BRCA2 gene; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Bone Marrow; CHEK2 gene; Cancer Patient; Cells; Cellular biology; Charge; Chromatin; Chronic; Cisplatin; Clinical; Clonal Expansion; DNA; DNA Binding; DNA Damage; DNA Ligases; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA lesion; Defect; Development; Dose; Dysplasia; Embryo; Erythropoiesis; Flow Cytometry; Fluorescence; Genes; Genetic Epistasis; Goals; Hematopoiesis; Hematopoietic; Histones; Hypersensitivity; Impairment; In Vitro; Knock-in; Knockout Mice; Lasers; Ligase; Ligation; Link; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mediating; Modeling; Molecular Biology; Mus; Mutation; Myelogenous; Okazaki fragments; Patients; Photobleaching; Physical condensation; Poly Adenosine Diphosphate Ribose; Proteins; Relaxation; Risk; Role; S phase; Site; Specificity; Splenomegaly; TP53 gene; Testing; Toxic effect; Withdrawal; XRCC1 gene; base; brca gene; cancer cell; cancer therapy; chemotherapy; effective therapy; in vivo; inhibitor; live cell imaging; malignant breast neoplasm; mouse genetics; mouse model; novel; premature; prevent; recruit; repaired

PROJECT SUMMARY/ABSTRACT PARP1 and PARP2 are DNA damage-induced poly-ADP-ribose (PAR) transferases, which are recruited to and activated by DNA breaks. Active PARP1&2 PARylate themselves and histones to promote DNA repair and chromatin relaxation. Dual-specificity inhibitors (PARPi) for PARP1 and PARP2 are currently used for the treatment of BRCA1/2-deficient breast, ovarian, pancreatic, and prostate cancers. However, severe anemia occurs in ~ one-third of patients, leading to dose reduction and premature termination of PAPRi therapy. PARPi also cause significant clonal hematopoiesis, a condition that increases the risk for myeloid dysplasia and acute- myeloid leukemia (MDS/AML). These hematopoietic toxicities are unexpected since the complete loss of Parp1 that is responsible for >80% of DNA damage-induced PARylation in cells, has no impact on hematopoiesis. To understand this potential on-target toxicity of PARPi, we generated mouse models with knockin catalytically inactive mutations in Parp2 – Poly-ADP-Ribosylation (PARylation) deficient (E534A, Parp2EA) or Mono-ADP- Ribosylation (MARylation) deficient (H404A, Parp2HA). In contrast to the normal development of Parp2-/- mice, mice expressing PARylation deficient PARP2 (Parp2EA/EA) died at embryonic day 16.5 (E16.5) with severe anemia and stage-specific blocks in erythropoiesis. The Parp2HA/HA mice are viable but display splenomegaly and chronic anemia. Meanwhile, our cell biology analyses suggest that clinically used PARPi effectively stalls PARP2, but not PARP1, on laser-induced DNA damage sites. Based on these and other observations, we hypothesize that PARP2 has PARylation-dependent structural functions that preferentially block nick ligation during DNA replication, underlie the PARP inhibitors-induced erythropoiesis defects and anemia. Specifically, we propose that catalytically inactive PARP2 were trapped on DNA breaks, especially 5’pho-nicks, where they prevent other repair factors (e.g., Ligase1) from accessing the DNA nicks. Correspondingly, like the Parp2EA/EA mice, Lig1-/- mice also succumbed to lethal anemia at E16.5. To test our hypothesis, we will Aim 1) investigate the mechanism that regulates PARP2 recruitment and dynamics at DNA damage sites using live-cell imaging and bulk biochemical assays; Aim 2) determine the impacts of catalytically inactive PARP2 on the recruitment and function of other DNA repair factors, and DNA replication in vivo and in vitro; Aim 3) characterize the mouse models expressing catalytically inactive - Parp2 and investigate how the loss of Trp53, CHK2, two genes associated clonal hematopoiesis, modulates the anemia in Parp2EA/EA models. The completion of the proposal will characterize the previously unrecognized structural functions of PARP2, address how catalytically inactive PARP2 compromises DNA replication and selectively abrogates erythropoiesis, providing the strategy to mitigate this on-targeted toxicity of PARP inhibitors by reducing PARP2 stalling.

项目总结/摘要 PARP 1和PARP 2是DNA损伤诱导的多聚ADP核糖（PAR）转移酶，其被募集 并被DNA断裂激活活性PARP 1和2 PARylate自身和组蛋白以促进DNA修复 和染色质松弛。PARP 1和PARP 2的双特异性抑制剂（PARPi）目前用于治疗糖尿病。 治疗BRCA 1/2缺陷的乳腺癌、卵巢癌、胰腺癌和前列腺癌。然而，严重贫血 在约三分之一的患者中发生，导致剂量减少和PAPRi治疗的过早终止。PARPi 也会导致显著的克隆性造血，这种情况会增加骨髓发育不良和急性- 骨髓性白血病（MDS/AML）。这些造血毒性是出乎意料的，因为Parp 1的完全丧失 其负责细胞中>80%的DNA损伤诱导的PAR化，对造血没有影响。到 为了理解PARPi的这种潜在的靶向毒性，我们用催化敲入产生了小鼠模型， Parp 2-聚ADP-核糖基化（PAR化）缺陷（E534 A，Parp 2 EA）或单ADP-核糖基化（PAR化）缺陷（E534 A，Parp 2 EA）中的无活性突变 核糖基化（MAR化）缺陷（H404 A，Parp 2 HA）。与Parp 2-/-小鼠的正常发育相反， 表达PAR化缺陷型PARP 2（Parp 2 EA/EA）的小鼠在胚胎第16.5天（E16.5）死亡， 贫血和红细胞生成阶段特异性阻滞。Parp 2 HA/HA小鼠存活，但显示脾肿大 和慢性贫血。同时，我们的细胞生物学分析表明，临床使用的PARPi有效地阻止了 PARP 2，而不是PARP 1，在激光诱导的DNA损伤位点。根据这些和其他观察，我们 假设PARP 2具有优先阻断切口PAR化依赖性结构功能 DNA复制过程中的连接，是PARP介导的红细胞生成缺陷和贫血的基础。 具体地说，我们提出，无催化活性的PARP 2被困在DNA断裂，特别是5 'pho-nicks， 其中它们阻止其它修复因子（例如，连接酶1）进入DNA缺口。相应地，如 Parp 2 EA/EA小鼠、Lig 1-/-小鼠也在E16.5死于致死性贫血。为了验证我们的假设，我们将目标1） 研究使用活细胞在DNA损伤位点调节PARP 2募集和动力学的机制 成像和大量生物化学测定;目的2）确定无催化活性的PARP 2对细胞的影响， 其他DNA修复因子的募集和功能，以及体内和体外的DNA复制;目的3）表征 表达无催化活性的-Parp 2的小鼠模型，并研究Trp 53，CHK 2，两个 与克隆造血相关的基因调节Parp 2 EA/EA模型中的贫血。的完成 一项提案将描述PARP 2以前未被认识到的结构功能， 失活的PARP 2损害DNA复制并选择性地消除红细胞生成， 以通过减少PARP 2停滞来减轻PARP抑制剂的这种靶向毒性。